This API-20E test strip (from bioMerieux, Inc.) is used to identify the enteric gram
negative rods (although API makes a variety of other test strips for yeast, Staph, ...
API 20E
This API-20E test strip (from bioMerieux, Inc.) is used to identify the enteric gram negative rods (although API makes a variety of other test strips for yeast, Staph, anaerobes, etc.) 20 separate test compartments are on the strip, all dehydrated. A bacterial suspension is used to rehydrate each of the wells. Some of the wells will have color changes due to pH differences: others produce end products that have to be identified with reagents. A profile number is determined from the sequence of + and - test results, then looked up in a codebook having a correlation between numbers and bacterial species.
OBJECTIVE: Learn how to perform and interpret the miniaturized, multi-test technique for bacterial identification.
MATERIALS NEEDED: agar plates of bacterial species 0.85% NaCl solutions (5ml) sterile Pasteur pipettes + bulbs sterile mineral oil API 20E test strip (for oxidase - gram negative rods) API test strip incubation chamber AFTER INCUBATION: 10% FeCl3, Barrett's reagents A and B, Kovac's reagent PROCEDURE: Prepare a suspension of the bacteria in the saline tube 1. Inoculate a large colony (2-3mm diameter)of the bacterium (pure culture) into the 0.85% NaCl solution, making sure that the suspension is homogenous and without clumps of floating bacteria. 2. Use a McFarland barium sulfate standard #3 to quantitate the suspension and to produce a standard inoculums size.. Inoculate the API strip 1. Holding the strip at a slight angle up from the table top, you will now inoculate the bacterial suspension into each well with the sterile pipette.
2. Touch the end of the pipette to the side of the cupule, allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. This should eliminate any bubbles forming in the wells. Each well should be filled up to the neck (see diagram). 3. CIT, VP, and GEL have boxes around their names. These test wells will be filled all the way up to the top of the well. 4. LDC, ODC, ADH, H2S, and URE are filled as described in step B, but they will then be filled up to the top with sterile mineral oil. Incubate the strip in its chamber 1. The bottom of the incubation chamber has small indented wells in the bottom: fill it with water just enough to fill these indentations. 2. Place the strip into this bottom. There should not be so much water that it slops onto the API strip. 3. Place the top of the incubation chamber over the bottom, and label it. 4. Place the strip at 37o C for 18-24 hours. INTERPRETATION: 1. Add the proper reagents to the compartments: o 1 drop of Kovac's to the IND (read within a couple of minutes) o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes) o 1 drop of FeCl3 to TDA 2. Read all other tests as described (chart below) without reagents using the table below.. 3. Record results on the diagram handed out to you in lab. The oxidase test reaction should be negative, and is added as the last test result. 4. Take your result sheet to the computer to INPUT YOUR REACTIONS: The API 20E website database will provide you with potential organisms. A more specific 9-digit number can be obtained with a few more tests:, but NOT DURING THIS LAB. (pictures from the API 20E documentation, from BioMerieux)
READING THE API 20 TESTS
SUBSTRATE
REACTION TESTED
- RESULTS
+ RESULTS
ONPG
ONPG
beta-galactosidase
colorless
yellow
ADH
arginine
arginine dihydrolase
yellow
red/orange
LDC
lysine
lysine decarboxylase
yellow
red/orange
ODC
ornithine
ornithine decarboxylase
yellow
red/orange
CIT
citrate
citrate utilization
pale green/yellow
blue-green/blue
H2S
Na thiosulfate
H2S production
colorless/gray
black deposit
URE
urea
urea hydrolysis
yellow
red/orange
TDA
tryptophan
deaminase
yellow
brown-red
IND
tryptophan
indole production
yellow
red (2 min.)
VP
Na pyruvate
acetoin production
colorless
pink/red (10 min.)
GEL
charcoal gelatin
gelatinase
no diffusion of black
black diffuse
GLU
glucose
fermentation/oxidation
blue/blue-green
yellow
MAN
mannitol
fermentation/oxidation
blue/blue-green
yellow
INO
inositol
fermentation/oxidation
blue/blue-green
yellow
SOR
sorbitol
fermentation/oxidation
blue/blue-green
yellow
RHA
rhamnose
fermentation/oxidation
blue/blue-green
yellow
SAC
sucrose
fermentation/oxidation
blue/blue-green
yellow
MEL
melibiose
fermentation/oxidation
blue/blue-green
yellow
AMY
amygdalin
fermentation/oxidation
blue/blue-green
yellow
ARA
arabinose
fermentation/oxidation
blue/blue-green
yellow
OX
oxidase
oxidase
colorless/yellow
violet
QUESTIONS: 1. What is the purpose of the water in the tray? 2. What is the function of the mineral oil? 3. What are the advantages of this test (compared to regular biochemical tube media)? 4. What are the disadvantages of this test?
LAB MANUAL: TABLE OF CONTENTS 8/2009, Jackie Reynolds, Richland College
